Heterogeneity of Monoclonal Antibodies

HONGCHENG LIU,1GEORGEEN GAZA-BULSECO,1DINESH FALDU,2CHRIS CHUMSAE,1JOANNE SUN1

1Process Sciences Department,Abbott Bioresearch Center,100Research Drive,Worcester,Massachusetts01605 2Quality Control Department,Abbott Bioresearch Center,100Research Drive,Worcester,Massachusetts01605

Received14May2007;revised2July2007;accepted31July2007

Published online in Wiley InterScience().DOI10.1002/jps.21180

ABSTRACT:Heterogeneity of monoclonal antibodies is common due to the various

modifications introduced over the lifespan of the molecules from the point of synthesis

to the point of complete clearance from the subjects.The vast number of modifications

presents great challenge to the thorough characterization of the molecules.This

article reviews the current knowledge of enzymatic and nonenzymatic modifications of

monoclonal antibodies including the common ones such as incomplete disulfide bond

formation,glycosylation,N-terminal pyroglutamine cyclization,C-terminal lysine

processing,deamidation,isomerization,and oxidation,and less common ones such

as modification of the N-terminal amino acids by maleuric acid and amidation of the

C-terminal amino acid.In addition,noncovalent associations with other molecules,

conformational diversity and aggregation of monoclonal antibodies are also discussed.

Through a complete understanding of the heterogeneity of monoclonal antibodies,

strategies can be employed to better identify the potential modifications and thoroughly

characterize the molecules.ß2007Wiley-Liss,Inc.and the American Pharmacists Association

J Pharm Sci97:2426–2447,2008

Keywords:Structure;Stability;Protein aggregation;Glycosylation;Deamidation;

Analytical biochemistry

INTRODUCTION

The primary structure of an antibody from a single cell clone is homogeneous.However,all the monoclonal antibodies studied are hetero-geneous in nature.It has been generally recognized that heterogeneity,especially charge hetero-geneity,is a common feature of monoclonal antibodies including monoclonal antibodies of different species such as human,1–11rabbit,12,13 mouse,14–28chimeric mouse/human,29,30humaniz-ed and fully human31–48recombinant monoclonal antibodies.

In1970,Awdeh et al.4carried out an interesting experiment using human tumor cell line5563,which synthesizes an IgG2a antibody.They observed a single homogeneous population of this antibody on isoelectric focusing gel electrophoresis(IEF) when pulse-labeling with radio-labeled amino acids was only carried out for10min.Soon after, the homogeneous population rapidly became hetero-geneous.It has been realized that enzymatic and nonenzymatic modifications including formation of disulfide bonds,glycosylation,N-terminal gluta-mine cyclization,C-terminal lysine processing, deamidation,oxidation,glycation,and peptide bond cleavage can cause heterogeneity.Depending on the type of

Correspondence to:Hongcheng Liu(Telephone:508-849-

2591;Fax:508-793-4885;E-mail:Hongcheng.liu@)

Journal of Pharmaceutical Sciences,Vol.97,2426–2447(2008)

ß2007Wiley-Liss,Inc.and the American Pharmacists Association

2426JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008